Aurora kinase inhibitors

ABSTRACT

The invention provides a compound of formula I: or a salt thereof, wherein ring A and R 1 -R 4  have any of the values defined in the specification. The compounds have activity as Aurora B kinase inhibitors and are useful for treating conditions associated with Aurora B kinase activity (e.g. cancer).

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of priority of U.S.application Ser. No. 61/719,215, filed Oct. 26, 2012, which applicationis herein incorporated by reference.

GOVERNMENT FUNDING

This work was supported by The Hormel Foundation and National Institutesof Health grants R37 CA081064, CA120388, ES016548, CA0227501 andNational Cancer Institute Contract No. HHSN-261200533001C-NO1-CN-53301.The United States Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Targeting the progression of mitosis is a highly successful strategy foranticancer treatment (Jackson, J. R., et al., Nat Rev Cancer, 2007. 7(2): p. 107-17). A closely related subgroup of three serine/threoninekinases, the Aurora kinases, are believed to play a key role in proteinphosphorylation in mitosis and have been shown to contribute in thedevelopment and progression of cancer. In mammals, the Aurora kinasefamily comprises three members: Aurora A, B and C (Carmena, M. and W. C.Earnshaw, Nat Rev Mol Cell Biol, 2003. 4 (11): p. 842-54). They displaydistinct roles during mitosis, which are reflected in their subcellularlocations and functions. Aurora A is localized at the centrosome fromthe time of centrosome duplication through mitotic exit. It has beenimplicated in several processes required for the generation of bipolarspindle apparatus, including centrosome maturation and separation(Andrews, P. D., Oncogene, 2005. 24 (32): p. 5005-15; and Barr, A. R.and F. Gergely, J Cell Sci, 2007. 120 (Pt 17): p. 2987-96). Smallmolecule inhibition of Aurora A kinase activity causes defects incentrosome separation with the formation of characteristic monopolarspindles (Marumoto, T., D. Zhang, and H. Saya, Nat Rev Cancer, 2005. 5(1): p. 42-50). Aurora B is localized to the centromeres from theprophase to the metaphase-anaphase transition. Thereafter, it islocalized to midzone spindle microtubules during the telophase andsubsequently to midbody during cytokinesis. Aurora B is a chromosomalpassenger protein in complex with the inner centromere proteins(INCENP), survivin, and borealin. During mitosis, as the“equatorial-kinase”, Aurora B is required for histone H3phosphorylation, chromosome bi-orientation, the spindle assemblycheckpoint, and cytokinesis (Kallio, M. J., et al., Curr Biol, 2002. 12(11): p. 900-5; and Carvajal, R. D., A. Tse, and G. K. Schwartz, ClinCancer Res, 2006. 12 (23): p. 6869-75). Inhibition of Aurora B kinaseactivity with small molecules leads to failure in cytokinesis andabnormal exit from mitosis, resulting in endoreduplication, polyploidycells, and ultimately apoptosis (Hauf, S., et al., J Cell Biol, 2003.161 (2): p. 281-94; and Ditchfield, C., et al., J Cell Biol, 2003. 161(2): p. 267-80). Aurora C is also a chromosomal passenger proteinconsidered to have a similar sub-cellular location as Aurora B. It hasbeen described only in mammals, where it is expressed in testis andcertain tumor cell lines and localizes to spindle poles during latemitosis (Carmena, M. and W. C. Earnshaw, Nat Rev Mol Cell Biol, 2003. 4(11): p. 842-54; and Chen, H. L., et al., J Biomed Sci, 2005. 12 (2): p.297-310).

Inhibition of Aurora kinases had been shown to be an effective strategyfor anticancer therapy, and several Aurora inhibitors have beendescribed, including VX-680 (Harrington, E. A., et al., Nat Med, 2004.10 (3): p. 262-7), Hesperadin (Hauf, S., et al., J Cell Biol, 2003. 161(2): p. 281-94), AZD1152 (Yang, J., et al., Blood, 2007. 110 (6): p.2034-40), and MLN8237 (Gorgun, G., et al., Blood, 2010. 115 (25): p.5202-13.). More than 30 small molecule Aurora kinase inhibitors arecurrently in different stages of preclinical and clinical development(Kollareddy, M., et al., Invest New Drugs, 2012), however, none have yetbeen approved by the FDA for clinical use.

Currently there is a need for agents that are useful for treating orpreventing cancer.

SUMMARY OF THE INVENTION

A series of compounds have been identified that possess activity asAurora B kinase inhibitory properties. Accordingly the inventionprovides a compound of the invention which is a compound of formula I:

wherein:

ring A is optionally substituted with one or more groups independentlyselected from hydroxy, halo, cyano, nitro, (C₁-C₆)alkyl,(C₃-C₆)cycloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, methylenedioxy, ethylenedioxy, or(C₂-C₆)alkanoyloxy, wherein any (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl,(C₁-C₆)alkoxy is optionally substituted with one or more halo;

R¹ is H, halo, cyano, nitro, (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, methylenedioxy,ethylenedioxy, or (C₂-C₆)alkanoyloxy, wherein any (C₁-C₆)alkyl,(C₃-C₆)cycloalkyl, (C₁-C₆)alkoxy is optionally substituted with one ormore halo; or R¹ and R² taken together are methylenedioxy orethylenedioxy;

R² is H, halo, cyano, nitro, (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, methylenedioxy,ethylenedioxy, or (C₂-C₆)alkanoyloxy, wherein any (C₁-C₆)alkyl,(C₃-C₆)cycloalkyl, (C₁-C₆)alkoxy is optionally substituted with one ormore halo; or R¹ and R² taken together are methylenedioxy orethylenedioxy; or R² and R³ taken together are methylenedioxy orethylenedioxy;

R³ is H, halo, cyano, nitro, (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, methylenedioxy,ethylenedioxy, or (C₂-C₆)alkanoyloxy, wherein any (C₁-C₆)alkyl,(C₃-C₆)cycloalkyl, (C₁-C₆)alkoxy is optionally substituted with one ormore halo; or R² and R³ taken together are methylenedioxy orethylenedioxy; or R³ and R⁴ taken together are methylenedioxy orethylenedioxy; and

R⁴ is H, halo, cyano, nitro, (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, methylenedioxy,ethylenedioxy, or (C₂-C₆)alkanoyloxy, wherein any (C₁-C₆)alkyl,(C₃-C₆)cycloalkyl, (C₁-C₆)alkoxy is optionally substituted with one ormore halo; or R³ and R⁴ taken together are methylenedioxy orethylenedioxy;

or a salt thereof.

The invention also provides a pharmaceutical composition comprising acompound of formula I or a pharmaceutically acceptable salt thereof, anda pharmaceutically acceptable excipient.

The invention also provides a method for treating or preventing cancerin an animal comprising administering a compound of formula I or apharmaceutically acceptable salt thereof to the animal.

The invention also provides a compound of formula I or apharmaceutically acceptable salt thereof for use in medical therapy.

The invention also provides a compound of formula I or apharmaceutically acceptable salt thereof for the prophylactic ortherapeutic treatment of cancer.

The invention also provides a method of inhibiting Aurora B kinase in ananimal in need of such treatment comprising administering a compound offormula I, or a pharmaceutically acceptable salt thereof, to the animal.

The invention also provides a method of treating a pathologicalcondition associated with Aurora B kinase in an animal comprisingadministering an effective Aurora B kinase inhibitory amount of acompound of formula I, or a pharmaceutically acceptable salt thereof, tothe animal.

The invention also provides a compound of formula I, or apharmaceutically acceptable salt thereof, for the prophylactic ortherapeutic inhibition of Aurora B kinase.

The invention also provides a compound of formula I, or apharmaceutically acceptable salt thereof, for the prophylactic ortherapeutic treatment of a pathological condition associated with AuroraB kinase

The invention also provides the use of a compound of formula I, or apharmaceutically acceptable salt thereof to prepare a medicament forinhibiting Aurora B kinase in an animal.

The invention also provides the use of a compound of formula I, or apharmaceutically acceptable salt thereof, to prepare a medicament fortreating a pathological condition associated with Aurora B kinase in ananimal.

The invention also provides processes and intermediates disclosed hereinthat are useful for preparing a compound of formula I or a salt thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. (A) Compounds 1-8 inhibit Aurora B kinase activity in vitro. Aninactive histone 3.3 protein was used as the substrate for an in vitrokinase assay with active Aurora B and 100 μmol/L ATP. Proteins wereresolved by Western blotting. (B) Shows the percent of Aurora Bremaining kinase activity.

FIG. 2. Compounds 1-8 decrease viability of HCT116 (upper panel) andHT29 (lower panel) colon cancer cells. At 5 μM, each of the compoundscauses a significant decrease in viability of colon cancer cells.

FIGS. 3A and 3B. Compounds 1-8 suppress the anchorage-independent growthof HCT116 cells. The asterisk indicates a significant (*p<0.05,**p<0.01) decrease in colony formation in cells treated with eachcompound compared with the DMSO treated control group. Twoconcentrations of each compound, 0.5 μM and 5 μM were tested. At 0.5 μMcompounds 1, 5, 6 and 8 showed the best efficiency, followed bycompounds 3 and 7. Compounds 2 and 4 had no effect. At 5 μM, all thecompounds were highly effective to inhibit anchorage-independent growth.

FIGS. 4A and 4B. Compounds 1-8 suppress the anchorage-independent growthof HT29 colon cancer cells at a concentration of 5 μM. The asteriskindicates a significant (**p<0.01) decrease in colony formation in cellstreated with each compound compared with the DMSO-treated group.

FIG. 5. Illustrates Xenograph data from Example 5 for Compound 1.

DETAILED DESCRIPTION

The following definitions are used, unless otherwise described: halo isfluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc. denote both straightand branched groups; but reference to an individual radical such aspropyl embraces only the straight chain radical, a branched chain isomersuch as isopropyl being specifically referred to.

As used herein, “animal” includes a mammal such as for example, a human.

As used herein “pathological condition associated with Aurora B kinase”includes, cancer.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase.

When a bond in a compound formula herein is drawn in anon-stereochemical manner (e.g. flat), the atom to which the bond isattached includes all stereochemical possibilities. When a bond in acompound formula herein is drawn in a defined stereochemical manner(e.g. bold, bold-wedge, dashed or dashed-wedge), it is to be understoodthat the atom to which the stereochemical bond is attached is enrichedin the absolute stereoisomer depicted unless otherwise noted. In oneembodiment, the compound may be at least 51% the absolute stereoisomerdepicted. In another embodiment, the compound may be at least 60% theabsolute stereoisomer depicted. In another embodiment, the compound maybe at least 80% the absolute stereoisomer depicted. In anotherembodiment, the compound may be at least 90% the absolute stereoisomerdepicted. In another embodiment, the compound may be at least 95 theabsolute stereoisomer depicted. In another embodiment, the compound maybe at least 99% the absolute stereoisomer depicted.

Specific values listed below for radicals, substituents, and ranges, arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents.

Specifically, (C₁-C₆)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;(C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl; (C₁-C₆)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy,butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy;(C₁-C₆)alkanoyl can be acetyl, propanoyl or butanoyl(C₁-C₆)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, orhexyloxycarbonyl; and (C₂-C₆)alkanoyloxy can be acetoxy, propanoyloxy,butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy.

A specific compound of formula I is a compound of formula Ia:

A specific value for ring A is substituted with one or more(C₁-C₆)alkoxy.

A specific value for ring A is 3,4,5-trihydroxyphenyl or 4-nitrophenyl.

A specific compound is a compound wherein at least one of R¹, R², R³,and R⁴ is other than H.

A specific compound is a compound wherein at least one of R¹, R², R³,and R⁴ is halo, cyano, nitro, (C₃-C₆)cycloalkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, or (C₂-C₆)alkanoyloxy.

A specific compound is a compound wherein at least one of R¹, R², R³,and R⁴ is halo.

A specific compound is a compound wherein at least one of R¹, R², R³,and R⁴ is fluoro or chloro.

A specific compound is a compound wherein R¹, R², R³, and R⁴ are each H.

A specific value for R² is halo.

A specific value for R² is fluoro or chloro.

A specific value for R³ is halo.

A specific value for R³ is fluoro or chloro.

Processes for preparing compounds of formula I are provided as furtherembodiments of the invention and are illustrated in Scheme 1 in whichthe meanings of the generic radicals are as given above unless otherwisequalified.

In cases where compounds are sufficiently basic or acidic, a salt of acompound of formula I can be useful as an intermediate for isolating orpurifying a compound of formula I. Additionally, administration of acompound of formula I as a pharmaceutically acceptable acid or base saltmay be appropriate. Examples of pharmaceutically acceptable salts areorganic acid addition salts formed with acids which form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartrate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The compounds of formula I can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of formula Ito the skin are known to the art; forexample, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

The ability of a compound of the invention to inhibit Aurora B kinase orto treat cancer may be determined using pharmacological models which arewell known to the art, or using the assays described in the Examplesbelow.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES Example 1 Preparation of Compound 1.

To a stirred solution of isatin (10 mmol) and diethylamine (0.3 mmol) inethanol was added 3,4-(Methylenedioxy)benzylideneacetone (10 mmol). Theresulting mixture was stirred at room temperature for 12 h. The solidseparated out was filtered and washed with ether and ethyl acetate. Theobtained yellow solid was recrystallized from ethanol to give 2.8 g ofA1. 1H NMR (400 MHz, d₆-DMSO): 10.21 (s, 1H), 7.45 (d, 1H, J=16.05),7.30 (s, 1H), 7.25 (d, 1H, J=7.44 Hz), 7.13-7.18 (m, 2H), 6.95 (d, 1H,J=8.04 Hz), 6.87 (t, 1H, J=7.44 Hz), 6.75 (d, 1H, J=7.64 Hz), 6.61 (d,1H, J=16.24 Hz), 6.07 (s, 2H), 6.01 (s, 1H), 3.60 (d, 1H, J=16.44 Hz),3.18 (d, 1H, J=16.44 Hz).

To a stirred suspension of A1 (0.5 g) in ethanol (15 mL) was added 37%HCl (5 mL). The reaction mixture was stirred at room temperature for 7days or heat at 80° C. for 30 min. After this time, the obtained redsolid (compound 1) was filtered and purified by recrystallization fromethanol. 1H NMR (400 MHz, d₆-DMSO): 10.74 (s, 1H), 8.35 (d, 1H, J=7.64Hz), 7.70 (d, 1H, J=16.05 Hz), 7.49 (d, 1H, J=1.37 Hz), 7.31-7.37 (m,3H), 7.25 (s, 1H), 6.95-7.02 (m, 2H), 6.87 (d, 1H, J=7.83 Hz), 6.11 (s,2H).

Example 2 Preparation of Compound 4.

Compound 4 was prepared according to the method described in Example 1using 4-Chloroisatin (10 mmol) and3,4-(Methylenedioxy)benzylideneacetone (10 mmol).

Compound A2: 1H NMR (400 MHz, d₆-DMSO): 10.47 (s, 1H), 7.49 (d, 1H,J=16.24 Hz), 7.33 (s, 1H), 7.17 (m, 2H), 6.95 (d, 1H, J=8.02 Hz), 6.84(d, 1H, J=8.22 Hz), 6.75 (d, 1H, J=7.63 Hz), 6.63 (d, 1H, J=16.24 Hz),6.18 (s, 1H), 6.07 (s, 2H), 3.98 (d, 1H, J=17.02 Hz), 3.30 (d, 1H,J=17.02 Hz).

Compound 4: MS (ESI): Calculated: 353.0455 Obtained: 354.0047 (M+1)355.0081 (M+2) 356.0019 (M+3).

Example 3 Preparation of Compound 5

To a stirred solution of isatin (10 mmol) and diethylamine (0.3 mmol) inethanol was added 3,4,5-trimethoxybenzylideneacetone (10 mmol). Theresulting mixture was stirred at room temperature for 12 h. After thistime, the solution was evaporated and purified by column chromatographyto give A3. 1H NMR (400 MHz, d₆-DMSO): 10.22 (s, 1H), 7.50 (d, 1H,J=16.25), 7.26 (d, 1H, J=7.44 Hz), 7.15 (t, 1H, J=7.63 Hz), 7.01 (s,2H), 6.88 (t, 1H, J=7.43 Hz), 6.71-6.79 (m, 2H), 6.03 (s, 1H), 3.80 (s,6H), 3.68 (s, 3H), 3.63 (d, 1H, J=16.24 Hz), 3.18 (d, 1H, J=16.44 Hz).

To a stirred solution of A3 (50 mg) in ethanol (2 mL) was added 37% HCl(0.5 mL). The reaction mixture was stirred at room temperature for 3 h.After this period, the obtained red solid was filtered and purified byrecrystallization from ethanol to provide Compound 5. MS (ESI):Calculated: 365.1263; Obtained: 366.0841 (M+1).

Example 4 Preparation of Compound 7

Compound 7 was prepared according to the method described in Example 1using 5-fluoroisatin (10 mmol) and 4-Nitrobenzylidineacetone (10 mmol).

Compound A4: 1H NMR (400 MHz, d₆-DMSO): 10.29 (s, 1H), 8.25 (d, 2H,J=8.42 Hz), 7.94 (d, 2H, J=8.61 Hz) 7.65 (d, 1H, J=16.44), 7.20 (dd,1H), 6.90-7.02 (m, 2H), 6.75 (m, 1H), 6.21 (s, 1H), 3.72 (d, 1H, J=16.83Hz), 3.30 (d, 1H, J=16.83 Hz).

Compound 7: MS (ESI): Calculated: 338.0703 Obtained: 339.0292 (M+1).

Example 5 Preparation of Compound 8

Compound 8 was prepared according to the method described in Example 1using isatin (2 mmol) and 3-methoxy-4-tetrahydropyranoxybenzylidineacetone (or Vanillylidenacetone) (2 mmol). MS (ESI):Calculated: 321.1001; Obtained: 322.0638 (M+1).

Using procedures similar to those described herein the followingcompounds 2, 3, and 6 were also prepared. Compounds 1-8 shown in Table 1as well as salts thereof, are compounds of the invention.

TABLE 1 S. No. Structure MS (ESI) 1

Calculated: 319.0845; Obtained: 320.0902 (M + 1) 2

Calculated: 353.0455 Obtained: 354.0524 (M + 1) 355.0558 (M + 2);356.0497 (M + 3) 3

Calculated: 353.0455 Obtained: 354.0530 (M + 1) 355.0547 (M + 2);356.0485 (M + 3) 4

Calculated: 353.0455; Obtained: 354.0047 (M + 1), 355.0081 (M + 2), and356.0019 (M + 3) 5

Calculated: 365.1263; Obtained: 366.0841 (M + 1) 6

Calculated: 337.0750 Obtained: 338.0819 (M + 1) 7

Calculated: 338.0703; Obtained: 339.0292 (M + 1) 8

Calculated: 321.1001; Obtained: 322.0638 (M + 1).

The biological activity of representative compounds of the invention canbe evaluated using known assays or using the assays described in Example6.

Example 6 Biological Assays Reagents and Materials

All cell lines were purchased from American Type Culture Collection(ATCC) and were cultured in monolayers at 37° C. in a 5% CO₂ incubatoraccording to ATCC protocols. Cells were cytogenetically tested andauthenticated before the cells were frozen. Each vial of frozen cellswas thawed and maintained for about two months (10 passages). Fortransfection experiments, the jetPEI (Qbiogene, Inc., Montreal, Canada)transfection reagent was used following the manufacturer's instructions.

Anchorage-Independent Cell Transformation Assay

Tumor cells are suspended in basal medium Eagle (BME) medium and addedto 0.6% agar, with different concentrations of Compound 1 in a baselayer and a top layer of 0.3% agar. The cultures are maintained at 37°C. in a 5% CO₂ incubator for 1 to 2 weeks and then colonies are countedunder a microscope using the Image-Pro Plus software (v.4) program(Media Cybernetics, Silver Spring, Md.).

Cell Cycle and Apoptosis Analyses

Cells are plated in 60-mm plates and treated or not treated withCompound 1 for the indicated time. At each time point, cells are fixedin 70% ethanol and stored at −20° C. for 24 h. After staining, the cellcycle distribution or apoptosis is determined using a BD FACSCaliburFlow Cytometer (BD Biosciences, San Jose, Calif.).

MTS Assay

To estimate the cytotoxicity of Compound 1, cells are seeded (8×10³cells per well) in 96-well plates and cultured overnight. Cells are fedwith fresh medium and treated with different doses of Compound 1. Afterculturing for various times, the cytotoxicity of Compound 1 is measuredusing an MTS assay kit (Promega, Madison, Wis.) according to themanufacturer's instructions.

Western Blot Analysis

Proteins are resolved by SDS-PAGE and transferred onto polyvinylidenedifluoride membranes (Millipore, Mass.), which are blocked with nonfatmilk and hybridized with specific primary antibodies. The protein bandsare visualized using an enhanced chemiluminescence reagent (GEHealthcare, Pittsburgh, Pa.) after hybridization with a horseradishperoxidase-conjugated secondary antibody.

Aurora B and Aurora A In vitro Kinase Assay

Inactive histone 3 proteins (1 μg) are used as the substrate for an invitro kinase assay with 100 ng of active Aurora B or Aurora A kinase.Reactions are carried out in 1×kinase buffer (25 mM Tris-HCl pH 7.5, 5mM beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na₃VO₄, 10mM MgCl₂ and 5 mM MnCl₂) containing 100 μM ATP at 30° C. for 30 minutes.Reactions are stopped and proteins detected by Western blotting.

Immunofluorescence Microscopy

A549 cells are seeded in four-chamber slides and cultured overnight. Thecells are then treated with DMSO or Compound 1 (1 μM) for 48 h at 37° C.After treatment, the cells are washed with PBS and fixed with methanolfor 12 hours, followed by blocking with 3% PBS for 1 hour. The cells arethen incubated with α-tubulin antibodies (1:100) overnight and DNA isstained with 4′-6-diamidino-2-phenylindole (DAPI, Pierce) for 30 minutesat room temperature. The cells are evaluated by fluorescent microscopy.

Hematoxylin-Eosin Staining and Immunohistochemistry

Tumor tissues from mice are embedded in a paraffin block and subjectedto hematoxylin and eosin (H&E) staining and immunohistochemistry. Tumortissues are deparaffinized and hydrated, then permeabilized with 0.5%Triton X-100/l PBS for 10 min, hybridized with Ki-67 (1:500) as theprimary antibody and an HRP-conjugated goat anti-rabbit or mouse IgGantibody is used as the secondary antibody. After developing with3,30-diaminobenzidine, the sections are counterstained with H&E. Allsections are observed by microscope and the Image-Pro Plus software(v.4) program (Media Cybernetics).

Statistical Analysis

All quantitative data are expressed as mean values±S.D. or S.E. andsignificant differences are determined by Student's t test or by one-wayANOVA. A probability value of P<0.05 is used as the criterion forstatistical significance.

Results

The Predicted Binding Mode of Compound 1 with Aurora B and Cytotoxicity

A molecular docking analysis was performed using Glide v5.7 to screen acompound library against the structure of Aurora B. Compound 1 wasidentified as a potential Aurora B inhibitor based on its high dockingscore. The predicted binding mode of Compound 1 and Aurora B showed thatCompound 1 occupies the ATP-binding site and forms a hydrogen bond withamino acid Ala173 in the hinge linker region, which is quite similar tothe binding of other Aurora B kinase inhibitors. The toxicity ofCompound 1 on both MRC-5 normal lung cells and A549 lung cancer cellswas then examined. The result showed that Compound 1 possessedsubstantial toxicity to both cell types at concentrations greater than10 μM. At a concentration of 1 μM or less, no obvious cytotoxic effectswere observed in either cell line. At 5 μM, Compound 1 treatment for 48hours resulted in a weak toxicity toward A549 cancer cells, but not toMRC-5 normal cells.

Compound 1 Inhibits Anchorage-Independent Growth of Human Lung CancerCells

The effect of Compound 1 treatment on anchorage-independent growth ofhuman lung cancer cells, including A549, H1650, and H520 cells was thenexamined. Treatment of these cells with Compound 1 potently inhibitedthe anchorage-independent growth in a concentration-dependent manner.Compound 1 at 0.5 or 1 μM caused a decrease of more than 80% or 90%compared to control in all cell lines detected. The inhibition byCompound 1 was not due to cytotoxicity because no toxicity was observedat 1 μM Compound 1. Therefore, the results indicated that Compound 1 isa very potent compound possessing anti-tumor activity.

The effect of compound 1 in a pair of colon cancer cell lines, p53wildtype (HCT116 p53^(+/+)) and p53-deficient (HCT116 p53^(−/31) ) cellswas also examined to determine whether the sensitivity of cells tocompound 1 directly correlates with the status of p53 in cells. Softagar assay results showed that HCT116 p53^(−/31) cells are moresensitive to compound 1 than HCT116 p53^(+/+) cells. Compound 1 at 0.05μM caused a 40% inhibition of growth of HCT116 p53^(−/−) cells, whereasno inhibition was observed in the wildtype p53 cells. Moreover, 0.1 μMcompound 1 inhibited growth by more than 95% in HCT116^(−/−) cells butonly 55% inhibition in HCT116 p53^(+/+) cells. These data are consistentwith another report showing that p53^(−/−) cells are more sensitive toAurora B inhibitors than p53^(+/+) cells.

Compound 1 is a Potent Inhibitor of Aurora B, but not Aurora A

An in vitro kinase assay was used to determine whether Compound 1 couldinhibit Aurora B kinase activity using recombinant Aurora B protein andvarious concentrations of Compound 1. Results indicated that thephosphorylation of histone H3 on Ser10, an Aurora B substrate, wasstrongly inhibited by Compound 1 in a concentration-dependent manner.For example, 0.05 μM Compound 1 caused a 22% inhibition of Aurora Bkinase activity and 0.1 μM Compound 1 resulted in a 50% inhibition. At aconcentration of 1 μM, only a weak histone H (Ser10) band was observed.The effect of Compound 1 on Aurora A kinase activity using an in vitrokinase assay was also examined. No effect on histone H3 (Ser10)phosphorylation was observed at 1 μM Compound 1 compared with control.These results also demonstrate that compound 1 is a potent inhibitor ofAurora B kinase.

Compound 1 Blocks Phosphorylation of Histone H3 on Ser10 in Lung CancerCells

The effect of compound 1 on Aurora B downstream signaling was alsoevaluated to demonstrate that compound 1 is acting through inhibition ofAurora B in cancer cells. Evidence indicated that histone H3 is a directdownstream target of the Aurora kinases. Phosphorylation of a highlyconserved serine residue (Ser10) in histone H3 is thought to be crucialfor entry into mitosis. Compound 1 suppressed histone H3 phosphorylationon Ser10 in cancer cells in a dose- and time-dependent manner,suggesting that the Aurora B kinases are involved in the antitumoractivity of Compound 1.

Compound 1 Induces Polyploidy, Cell Cycle Arrest, and Apoptosis in LungCancer Cells

Aurora B inhibition leads to failure in cytokinesis and abnormal exitfrom mitosis, which could result in polyploidy cells, cell cycle arrest,and ultimately apoptosis. The ability to induce polyploidy cells wasfurther examined in A549 cells treated or not treated with Compound 1.Immunofluorescence results showed that treatment of A549 cells with 1 μMCompound 1 caused the induction of polyploidy cells, whereas nopolyploidy cells were observed in control cells. In addition, Compound 1treatment for 48 hours caused an increase in the number of A549 and H520cells occupying the G2/M phase. Moreover, exposure of these cells toCompound 1 for 72 hours induced apoptosis as measured by Annexin V/PIstaining. For example, exposure to 5 μM Compound 1 induced 48% or 82%apoptosis in A549 cells and H520 cells, compared to 8% or 38% inuntreated control cells, respectively. These results demonstrate thatcompound 1, as an Aurora B inhibitor, induces polyploidy, apoptosis andG2/M phase arrest in cancer cells and thus inhibits the growth of cancercells.

Knockdown of Aurora B Decreases the Sensitivity of Cancer Cells toCompound 1

The effect of knocking down Aurora B expression in A549 cancer cells onthe cell's sensitivity to Compound 1 was evaluated. The efficiency ofshRNA knockdown was examined and the expression of Aurora B wasobviously decreased after shRNA transfection. Moreover, the growth ofcells in soft agar also decreased after transfection compared with themock group. Compound 1 (0.5 μM) inhibited anchorage-independent growthof A549 cells transfected with mock shRNA by about 90%. In contrast, theinhibition was less than 40% in A549 cells transfected with Aurora BshRNA, indicating that A549 cells transfected with Aurora B shRNA weremore resistant to Compound 1 treatment. These results suggested thatAurora B plays an important role in the sensitivity of A549 cells to theanti-proliferative effects of Compound 1.

Kinase Profile Result

A kinase profile assay was performed by Millipore, to examine otherpotential targets of Compound 1. Only Akt2 activity was suppressed morethan 50% after 5 μM compound 1 treatment, compared with the control(Table 2). In addition, the kinase activity of Aurora A was decreasedless than 25% at 5 μM Compound 1.

TABLE 2 Kinase HOI-07 (5 μM) Abl 109 AMPK-alpha1 96 AMPK-alpha2 90Aurora A 76 CDK1/cyclinB 86 CDK2/cyclinA 98 CDK3/cyclinE 99 CHK2 75c-Kit 123 CSK 110 c-RAF 87 c-Src 99 DYRK2 97 EGFR 106 EGFR (T790M,L858R) 101 FAK 104 FGFR1 86 Flt3 92 Fyn 99 GSK3 alpha 85 GSK3 beta 94IGF-1R 105 IKK alpha 83 IKK beta 68 JNK1 alpha1 104 JNK2 alpha2 89 KDR93 LKB1 87 MAPK1 100 MAPK2 102 MEK1 104 Met 89 MKK4 130 MKK6 129 MKK7beta 94 MSK1 98 MSK2 79 mTOR 136 mTOR/FKBP12 86 p70S6K 72 PDK1 96 Pim-196 PKB alpha (Akt1) 68 PKB beta (Akt2) 47 RSK2 94 PI3-K alpha 102 PI3-Kbeta 103 PI3-K gamma 100 PI3-K delta 105Compound 1 Inhibits Akt1 and 2 Kinase Activities In vitro, but not in Exvivo

According to the kinase profiler assay results, Akt2 also might be apotential target of Compound 1. An Akt1 and Akt2 in vitro kinase assayswas performed. The results indicated that Compound 1 inhibited both Akt1and Akt2 in vitro kinase activities in a concentration-dependent manner.Treatment with 5 μM Compound 1 caused a 32% or 52% inhibition of Akt1 orAkt2 activity, respectively, which is quite consistent with the kinaseprofiler assay result. However, Western blot analysis of A549 cells andH520 cells treated with Compound 1 showed that Compound 1 treatment hasno effect on the phosphorylation of Akt or its downstream signalingmolecules, including Akt (Ser473), GSK3β (Ser9), p70S6K (Thr389) and S6(Ser235,236). These results indicated that although Compound 1 caninhibit Akt1 and Akt2 in vitro kinase activity, it has no effect on Aktactivity in cancer cells, suggesting that Akt1 and Akt2 might not be themajor antitumor targets of Compound 1.

Compound 1 Suppresses the Growth of A549 Xenografts In vivo

The ability of Compound 1 to inhibit the growth of human A549 lungcancer cell xenografts in athymic nude mice was then evaluated. Tumorvolumes were measured twice a week and mouse weights were determinedonce a week. Compound 1 caused a marked reduction in tumor size in thehuman A549 xenograft model. In mice treated with Compound 1 at 20 mg/kg,twice a week intraperitoneally, mean tumor volumes were reduced to 164mm³ in comparison with control group (408 mm³) (P<0.01). In addition, noobvious loss of body weight was observed, indicating that Compound 1 iswell tolerated by the mice. Moreover, the effects of Compound 1 on atumor proliferation marker were evaluated by immunohistochemistry andH&E staining of A549 tumor tissues after the 31 days of treatment. Theexpression of Ki-67 was markedly decreased by Compound 1. These resultsindicated that Compound 1 suppressed tumor growth in vivo.

Discussion

Cancer is a disease that is characterized by uncontrolled proliferationof abnormal cells. Modulation of atypical cell cycle regulation wouldtherefore be a valuable therapeutic strategy for different types oftumors. Aurora kinases regulate many processes during cell division.Aurora B kinases are essential for chromosome condensation, kinetochorefunction, cytokinesis and the proper function of the spindle-assemblycheckpoint when spindle tension is perturbed (Carmena, M. and W. C.Earnshaw, Nat Rev Mol Cell Biol, 2003. 4 (11): p. 842-54; Kallio, M. J.,et al., Curr Biol, 2002. 12 (11): p. 900-5; Ditchfield, C., et al., JCell Biol, 2003. 161 (2): p. 267-80; and Yang, H., et al., FEBS Lett,2005. 579 (16): p. 3385-91). Accumulating evidence has shown that AuroraB is implicated in cancer. For example, the expression of Aurora B isfrequently elevated in various types of cancer, including NSCLC, colon,prostate (Tatsuka, M., et al., Cancer Res, 1998. 58 (21): p. 4811-6;Vischioni, B., et al., Mol Cancer Ther, 2006. 5 (11): p. 2905-13; andChieffi, P., et al., Prostate, 2006. 66 (3): p. 326-33). The evidencelinking Aurora overexpression and malignancy has generated significantinterest in the development of small molecule inhibitors.

Oxindoles (indolin-2-ones) are an important class of molecules, whichare known to possess a wide variety of biological properties, and inparticular, as protein kinase inhibitors (Millemaggi A, T. R., EuropeanJournal of Organic Chemistry, 2010 (24): p. 4527-4547). In the presentstudy, Compound 1 was identified using molecular docking methods as anAurora B kinase inhibitor.

The results of an Aurora B kinase assay showed that Compound 1 potentlyand dose-dependently inhibited Aurora B kinase in vitro activity,indicating that this compound is a potent and novel Aurora B inhibitor.In addition, Compound 1 had no effect on Aurora A kinase in vitroactivity at the same concentration. A previous report showed that cellstreated with an Aurora kinase inhibitor entered and exited mitosiswithout cell division, and then proceeded to a second S phase.Therefore, the activity of Compound 1 on cancer cells was examined.Results showed that Compound 1 suppressed cell growth in a panel ofNSCLC cell lines, associated with induction of polyploidy cells,accumulation of G₂/M cells, as well as apoptosis, which is consistentwith Aurora B inhibition. Moreover, knocking down Aurora B expression inA549 cells decreased their sensitivity to Compound 1, indicating thatAurora B plays an important role in the antitumor activity ofCompound 1. An in vivo xenograft study also indicated that Compound 1effectively suppressed tumor growth without affecting mouse body weightand was accompanied with a decrease in Ki-67 expression, which is amarker of proliferation.

According to kinase profiling results, in which 49 kinases treated ornot treated with Compound 1, Akt might also be a potential target ofCompound 1. Further experimental results showed that Compound 1inhibited Akt1 and Akt2 kinase activity in vitro at a higherconcentration (1 μM or more) than that required for Aurora B inhibition(1 μM or lower). However, it had no effect on Akt downstream signalingin cancer cells, indicating that Akt might not be a major target ofCompound 1. Together, these results indicated that Compound 1 is apotent and selective inhibitor of Aurora B.

Example 7 Xenograft Mouse Model

Athymic nude mice (6 week old nu/nu female mice, Harlan Laboratory,Minneapolis, Minn.) were inoculated in the right flank with A549 lungcancer cells (3*10⁶ cells/mouse). Mice were maintained under “specificpathogen-free” conditions based on the guidelines established by theUniversity of Minnesota Institutional Animal Care and Use Committee.Tumors were allowed to grow to an average of ˜53.5±34.5 mm³ and thenmice were divided into 2 equal groups with a similar average tumorvolume (group 1; n=15), vehicle only; (group 2, n=20), compound 1 at 40mg/kg. Treatment with vehicle or compound 1 was initiated on Day 13after inoculation of cells and continued to Day 80 (˜9 wks) and wasadministered by oral gavage 5 times a week. Tumor volume was measuredonce a week and body weight was measured once a week. Compound 1 wasprepared in 2.5% DMSO/5% PEG 400/5% Tween-80 in 1×PBS and sonicated for˜20 minutes. The final tumors from the vehicle-treated group weresignificantly larger in volume (1004±140.1) than the Compound 1 treatedgroup (403.2±50.0). Data are shown as means±S.E (FIG. 1). No toxicitywas observed in any mice. Oral administration of Compound 1 was highlyeffective in preventing xenograft growth of A549 lung cancer cells.

Example 8

The following illustrate representative pharmaceutical dosage forms,containing a compound of formula I (‘Compound X’), for therapeutic orprophylactic use in humans.

(i) Tablet 1 mg/tablet Compound X = 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0

(ii) Tablet 2 mg/tablet Compound X = 20.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0500.0

(iii) Capsule mg/capsule Compound X = 10.0 Colloidal silicon dioxide 1.5Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0

(iv) Injection 1 (1 mg/ml) mg/ml Compound X = (free acid form) 1.0Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodiumchloride 4.5 1.0N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mg/ml) mg/ml Compound X = (free acid form) 10.0Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethyleneglycol 400 200.0 1.0N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(vi) Aerosol mg/can Compound X = 20.0 Oleic acid 10.0Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0Dichlorotetrafluoroethane 5,000.0The above formulations may be obtained by conventional procedures wellknown in the pharmaceutical art.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A compound of formula I:

wherein: ring A is substituted with one or more groups independentlyselected from hydroxy, halo, cyano, nitro, (C₁-C₆)alkyl,(C₃-C₆)cycloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, methylenedioxy, ethylenedioxy, or(C₂-C₆)alkanoyloxy, wherein any (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl,(C₁-C₆)alkoxy is optionally substituted with one or more halo; R¹ is H,halo, cyano, nitro, (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, methylenedioxy, ethylenedioxy,or (C₂-C₆)alkanoyloxy, wherein any (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl,(C₁-C₆)alkoxy is optionally substituted with one or more halo; or R¹ andR² taken together are methylenedioxy or ethylenedioxy; R² is H, halo,cyano, nitro, (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, methylenedioxy, ethylenedioxy,or (C₂-C₆)alkanoyloxy, wherein any (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl,(C₁-C₆)alkoxy is optionally substituted with one or more halo; or R¹ andR² taken together are methylenedioxy or ethylenedioxy; or R² and R³taken together are methylenedioxy or ethylenedioxy; R³ is H, halo,cyano, nitro, (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, methylenedioxy, ethylenedioxy,or (C₂-C₆)alkanoyloxy, wherein any (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl,(C₁-C₆)alkoxy is optionally substituted with one or more halo; or R² andR³ taken together are methylenedioxy or ethylenedioxy; or R³ and R⁴taken together are methylenedioxy or ethylenedioxy; and R⁴ is H, halo,cyano, nitro, (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, methylenedioxy, ethylenedioxy,or (C₂-C₆)alkanoyloxy, wherein any (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl,(C₁-C₆)alkoxy is optionally substituted with one or more halo; or R³ andR⁴ taken together are methylenedioxy or ethylenedioxy; or a saltthereof.
 2. The compound of claim 1 which is a compound of formula (Ia):

or a salt thereof.
 3. The compound of claim 1 wherein ring A issubstituted with one or more (C₁-C₆)alkoxy.
 4. The compound of claim 1wherein ring A is 3,4,5-trihydroxyphenyl or 4-nitrophenyl.
 5. Thecompound of claim 1 wherein at least one of R¹, R², R³, and R⁴ is otherthan H.
 6. The compound of claim 1 wherein at least one of R¹, R², R³,and R⁴ is halo, cyano, nitro, (C₃-C₆)cycloalkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, or (C₂-C₆)alkanoyloxy.
 7. Thecompound of claim 1 wherein at least one of R¹, R², R³, and R⁴ is halo.8. The compound of claim 1 wherein at least one of R¹, R², R³, and R⁴ isfluoro or chloro.
 9. The compound of claim 1 wherein R² is halo.
 10. Thecompound of claim 1 wherein R² is fluoro or chloro.
 11. The compound ofclaim 1 wherein R³ is halo.
 12. The compound of claim 1 wherein R³ isfluoro or chloro.
 13. A compound selected from:

and salts thereof.
 14. A pharmaceutical composition comprising acompound as described in claim 1 or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier.
 15. A method ofinhibiting Aurora B kinase in an animal in need of such treatmentcomprising administering a compound as described in claim 1 or apharmaceutically acceptable salt thereof to the animal.
 16. A method oftreating a pathological condition associated with Aurora B kinase in ananimal comprising administering an effective Aurora B kinase inhibitoryamount of a compound as described in claim 1 or a pharmaceuticallyacceptable salt thereof to the animal.
 17. A method of treating cancerin an animal comprising administering a compound as described in claim 1or a pharmaceutically acceptable salt thereof to the animal. 18-24.(canceled)